We have continued our studies of chromatin structure in the neighborhood of expressed genes. The globin gene family in chicken erythroid cells serves as a model system in which it is possible to study the mechanisms associated with regulation of the cluster and individual members of the family during erythroid development. We have focused attention on the 1.2 kb insulator DNA sequence at the 5' end of the chicken beta-globin locus, and elements upstream of it. This insulator is capable both of blocking the influence of outside enhancers and of preventing the encroachment of condensed chromatin that might shut down expression of the entire region. We showed that enhancer blocking activity is associated with binding of a single protein, CTCF, to a site within the enhancer. The insulator also has the separate ability to protect against position effects reporter genes that are stably transfected into cell lines or animals, serving as a boundary against encroachment of condensed chromatin. We found that this protective ability is present in a ?core' element, 250 bp long, from within the 1.2 kb insulator, and that deletion of subregions within the core that contain the CTCF site do not affect activity. However four other subregions corresponding to binding sites for nuclear proteins are important for position effect protection (boundary function). We have now identified several of the involved factors binding to these sites. One is a protein previously described in our laboratory, and which has a strong preference for oligo G sequences. It binds to two of the four sites. Other factors are associated with the other two sites. We have furthermore been able to detect binding at the insulator in vivo of co-factors that are known histone modification enzymes. These results are consistent with a model we have proposed in which barrier function is connected with multiple histone modifications in the neighborhood of the insulator. The globin insulator appears to serve as a barrier against encroachment of an upstream region of condensed chromatin about 16 kb in length. We have developed methods for analyzing the hydrodynamic properties of this upstream segment with high precision. In addition to measuring its sedimentation coefficient very accurately, we have also measured the buoyant density of this specific fragment. This allows us to calculate its frictional coefficient and leads to the conclusion that the particle shape is an extended rod, consistent with earlier structural proposals from a number of laboratories. This is important because vertebrate genomes have large numbers of interspersed condensed chromatin segments of this kind, and virtually nothing is known of their structure. We have also continued studies of the kinetics of transgene silencing, using our test construct which expresses a fragment of the IL2 receptor. Although in some gene systems DNA methylation is the primary event in gene silencing, we find here that histone H3 and H4 deacetylation occurs first, more or less simultaneously with loss of mRNA. However DNA methylation occurs much later and cannot be the cause of the silencing, but rather a consequence of other, earlier changes.